RATIONALE: Intracellular Ca stores play a critical role in signaling Ca2+ -induced keratinocyte differentia- tion. Keratinocyte endoplasmic reticulum (ER) Ca2+ stores are maintained by the Ca2+ ATPase ATP2A2, while a closely related Ca2+ATPase, ATP2C1, localizes to the Golgi and maintains the Ca2+ store in this organelle. Mutations in the ATP2A2 underlie the human skin condition Darier's disease (DD), characterized by incomplete keratinocyte differentiation. Work from ours and other laboratories demonstrates that the ATP2A2 and ATP2C1 act jointly to shape Ca2+ signaling stimulated by raised extracellular Ca2+, making keratinocytes the first mammalian cells known to be under dual control of both ER and Golgi Ca2+ stores. Our preliminary studies reveal that ATP2A2 dysfunction in DD keratinocytes is compensated, in part, by ATP2C1 upregulation. These compensatory changes allow DD keratinocytes to remain viable, since completely inactivating ATP2C1 in DD keratinocytes leads to cell death. However, compensatory ATP2C1 upregulation also leads to abnormally low cytosolic Ca2+ concentrations, and may underlie the unique pattern of abnormal differentiation seen in DD keratinocytes. In this proposal, we plan to focus on the molecular mechanisms by which ATP2A2 dysfunction and ATP2C1 upregulation lead to abnormal Ca2+-induced differentiation in DD keratinocytes. HYPOTHESIS: The impaired keratinocyte differentiation characteristic of DD results both from downstream abnormalities in Ca2+ signaling pathways controlled by the mutated ATP2A2 and by the compensatory changes in other Ca2+ signaling proteins, especially the ATP2C1 and plasma membrane ion channels. This abnormal differentiation can be normalized by correcting defects in Ca2+ homeostasis, applying other prodifferentiative agents that bypass the Ca2+-responsive pathways involved in involucrin synthesis, or a combination of these strategies. SPECIFIC AIMS: Aim#1: To Characterize the Ca2+ Signaling Defects and Adaptive Responses in DD Keratinocytes. Aim #2: To Determine Whether Impaired Ca2+ -induced Differentiation in DD is Caused by Abnormally Decreased Cytosolic Ca2+, Pathologic ERK Signaling Due to Abnormal Organelle Ca2+ Homeostasis, or Impaired SERCA2-dependent Nuclear Ca2+ Signaling Aim #3: To Normalize Differentiation in DD by Improving Ca2+ Homeostasis, Enhancing Involucrin Promoter Activation, or Both. The goal of this project is to define the pathogenic signaling pathways that lead to abnormal differentiation in Darier's disease, thus ameliorating the skin pathology of patients suffering from this condition.